This proposal investigates cell envelope stress responses in Bacillus subtilis, a model organism for the Gram-positive bacteria. Antibiotics and other agents that impair the synthesis or function of the cell membrane or peptidoglycan cell wall are of critical importance in clinical medicine. Exposure to sub-lethal levels of these antibiotics activates the transcription of large sets of genes. These cell envelope stress responses are frequently adaptive and include mechanisms that protect the organism against the antibiotics. In Bacillus subtilis, these stress responses are coordinated by several of the seven alternative sigma subunits for RNA polymerase of the extracytoplasmic function (ECF) subfamily. This proposal is focused on defining the role of ECF sigma factors in regulating gene expression and identifying pathways of adaptation and resistance to antibiotics. Using a combination of classical and molecular genetics, genomics, transcriptomics, and biochemical approaches, these studies will define the pathways that activate expression of the various ECF sigma factor regulons, identify genes and functions controlled by ECF sigma factors, and investigate novel pathways of antibiotic resistance. PUBLIC HEALTH RELEVANCE: The bacterial envelope, comprising a lipid membrane and a peptidoglycan cell wall, separates the cell from the environment and is a target for the majority of antibiotics currently in clinical use. This proposal studies how bacteria respond to antibiotics that block the synthesis or function of the cell envelope and addresses the mechanisms by which bacteria become resistant to antibiotics. This proposal focuses specifically on the model Gram-positive bacterium Bacillus subtilis, which is related to Staphylococcus aureus and pathogenic streptococcal and enterococcal strains.